Materials and methods for intracellular transport and their uses

ABSTRACT

Coupled polypeptides and fusion polypeptides for intracellular transport and their preparation and use, include (i) an aminoacid sequence with the transport function of herpesviral VP22 protein (or a homologue, e.g. from VZV, BHV or MDV) and (ii) another protein sequence selected from (a) proteins for cell cycle control; (b) suicide proteins; (c) antigenic sequences or antigenic proteins from microbial and viral antigens and tumour antigens; (d) immunomodulating proteins; and (e) therapeutic proteins. The coupled proteins can be used for intracellular delivery of protein sequences (ii), to exert the corresponding effector function in the target cell, and the fusion polypeptides can be expressed from corresponding polynucleotides, vectors and host cells.

FIELD OF THE INVENTION

[0001] The present invention concerns improvements, modifications anddevelopments in relation to transport proteins, intracellular transportand their applications. In particular embodiments, the invention relatesto fusion proteins comprising transport proteins comprising sequencesfrom herpesviral VP22 or from homologues or fragments thereof togetherwith sequences from other proteins; and to methods for their preparationand use. In particular embodiments, the invention relates to fusionproteins for cell cycle control, and to materials and methods for theirpreparation and their use. In particular examples the invention relatesto fusion proteins having both mammalian p53 functionality andherpesviral VP22 functionality. Other aspects of the invention will beapparent from the description and claims.

BACKGROUND OF THE INVENTION, AND PRIOR ART

[0002] Relevant to the present application is the inventors' own earlierinternational patent application WO 97/05255 (O'Hare and Elliott)(published after the priority date claimed for this application), whichrelates to VP22 protein and its properties and uses. Similarly theinventors' paper (Elliott and O'Hare (1997), in Cell, vol 88 pp 223-233(1997), relates to intercellular trafficking and protein delivery by aherpesvirus structural protein. Both these documents are herebyincorporated in their entirety by reference and made an integral part ofthis disclosure.

[0003] The inventors have shown that the HSV-1 virion protein VP22possesses an unusual intercellular trafficking mechanism, an effectparticularly described in specification WO 97/05265. VP22 is a 38 kDaprotein which in primary-expressing transfected mammalian cells islocated predominantly in the cytoplasm where it associates with cellularmicrotubules (see accompanying drawing, FIG. 1b), However a remarkableproperty of VP22 is its ability to spread throughout a monolayer ofnon-expressing cells. VP22 is transported from the cytoplasm anexpressing cell into neighbouring cells where it accumulates in thenucleus (FIG. 1b). The mechanism of this transport is still incompletelyunderstood, but has teen shown to be via a golgi-independent pathway andmay utilise the actin cytoskeleton. HIV-1 Tat (Ensoli et al., 1993,Fawell et. al., 1994) and a small number of other non-viral proteins(Jackson et al., 1992) have been attributed with intercellulartrafficking properties, but none appears to demonstrate this phenomenonas strikingly as VP22. A further important property of VP22 is that whenapplied exogenously to the medium of an untransfected cell monolayer, itcan be taken up by those untransfected cells where it accumulates in thecell nucleus.

[0004] The prior art generally includes a variety of antigens,immunomodulating proteins, proteins that are conditionally cytotoxic orlethal upon administration (to a cell containing them) of acorresponding drug or activator compound, proteins for cell cyclecontrol, and other therapeutic and diagnostic proteins, especially inthe forms of protein and polynucleotide sequences enabling geneticmanipulation by standard techniques. References to some examples ofthese materials are given below.

[0005] For example, among cell cycle control proteins, protein p53 isknown as a tumour suppressor. p53 is a 53kDa nuclear phosphoproprotein(FIG. 1c). Wild-type and mutant p⁵³ proteins have been expressed bymeans of recombinant vaccinia viruses, (Ronen et al., Nucleic AcidsResearch, 20:3435-3441, 1592). p53 functions to regulate cell cycleprogression and under conditions of DNA damage through a complex signaltransduction mechanism can induce cell cycle arrest or apoptosis (Levine1997). Failure to synthesize p53, or more commonly synthesis of amutated form of the protein can result in uncontrolled cellproliferation and tumour formation. It has been shown by several groupsthat exogenous addition of functional wild type p53 can promote cellcycle arrest and/or apoptosis resulting in tumour regression withexamples including cervical carcinomas (Hamada et al., 1999) and breastcancer xenografts (Nielsen et al., 1997). A number of p53 deliverysystems have been utilised in vivo and in vitro such as intravenousinjection of a p53:liposome complex (Kumar et at., 1997), directtransfection (Zheng et al., 1996) and adenoviral mediated transfer(Hamada et al. 1996, Sandig et al., 1997) but delivery of functionalprotein into a sufficiently high percentage of surviving cells remains adifficulty.

[0006] Also known from U.S. Pat. No. 5,434,710 (La Jolla: J C Reed etal) are regulatory elements linked to genes involved in cell death, asregulated by p53 tumour suppressor protein, and further proteins andtheir analogues or cell cycle control.

[0007] It remains desirable to provide particular further cell-deliveryconstructs for useful proteins.

SUMMARY AND DESCRIPTION OF THE INVENTION

[0008] According to an aspect of the present invention, there areprovided coupled proteins comprising transport protein sequencescomprising sequences from herpesviral VP22 or from homologues orfragments thereof, together with sequences from other proteins selectedfrom: (a) proteins for cell cycle control; (b) proteins that areconditionally cytotoxic or lethal upon administration (to a cellcontaining them) of a corresponding drug, pro-drug or activator compound(otherwise described herein as suicide proteins); (c) antigenicsequences or antigenic proteins (e.g. of greater than 12 aminoacidresidues in length) from microbial and viral antigens and tumourantigens; (d) immunomodulating proteins; and (e) therapeutic proteins.Examples of these kinds of proteins mentioned below. Thus, coupling orfusion to an aminoacid sequence with the transport function of VP22protein can provide a useful cell delivery construct for proteins of thekinds mentioned. (Where the context admits, ‘coupling products’ andsimilar expressions include reference to fusion proteins.)

[0009] Preferably the coupled proteins are fusion proteins, which canconveniently be expressed in known suitable host cells. Correspondingpolynucleotide sequences can be prepared and manipulated using elementsof per-se known and standard recombinant DNA technique and readilyavailable adaptations thereof. However, chemically-coupled products canfor certain applications be used if desired, and can be prepared fromthe individual protein components according to any of a variety ofper-se known chemical coupling techniques.

[0010] VP22 or a functional sub-sequence thereof, optionally with anadditional polypeptide tail for coupling, can be linked to otherproteins or nucleic acid by chemical coupling in any known suitablestandard manner.

[0011] Also provided by the invention are polynucleotides encoding thefusion proteins as described herein, including sequences correspondingto VP22 and another protein of one of the kinds mentioned above, andexpression cassettes, plasmids, vectors and recombinant cells comprisingthe polynucleotides. These can be formed and used in ways analogous toor readily adaptable from standard recombinant DNA technique. Thus,corresponding polynucleotides can encode a fusion polypeptide thatcomprises a sequence with the transport function of herpesviral VP22protein and a sequence with one of the functions specified herein. Thepolynucleotide can be comprised in an open reading frame operably linkedto a suitable promoter sequence, and can according to examples of theinvention form par, of an expression vector, e.g. comprising thepolynucleotide carried in a plasmid. The expression vector can be forexample a recombinant virus vector or a non-viral transfection vector.The vectors can for example be analogues or examples of those vectorsmentioned or described in WO97/05255, or of those mentioned or describedin WO 92/05263, WO 94/21807, or WO 96/26267. For nucleotide sequencethat are capable of being transcribed and translated to produce afunctional polypeptide, degeneracy of the genetic code results in anumber of nucleotide sequences that encode the same polypeptide. Theinvention includes all such sequences.

[0012] Thus products described herein can be used according to theinvention as transportable proteins capable of being taken up by atarget population of cells, e.g. so that an effector functioncorresponding to the polypeptide sequence coupled to the VP22, fromamong the kinds mentioned above, can take place within the target cellsthat have taken up the product. Thus, for example, the target cells maypresent desired tumour antigen epitopes in a case where the polypeptidesequence is from a chosen tumour antigen, or become subject to cellcycle control effects where the the polypeptide sequence is from a cellcycle control protein, or become in some degree susceptible to cellkilling or injury after additional treatment with a prodrug where thepolypeptide sequence is from a corresponding ‘suicide protein’. In use,many of the products described herein can be expressed as fusionproteins in a first part of the target population of cells, exportedtherefrom, and taken up by a second part of the target population ofcells not directly producing the protein, Also within the invention aremammalian and microbial host cells comprising such vectors or otherpolynucleotides encoding the fusion proteins, and their production anduse.

[0013] A fusion polypeptide as desired herein can be transported to atarget population of cells, by introducing a polynucleotide or othervector encoding the fusion polypeptide into a first part of the targetpopulation of cells, e.g. by transfection or microinjection; expressingthe encoding polynucleotide to produce the fusion polypeptide, therebyto cause it to be exported from said first part of said targetpopulation, and to cause it to be taken up by a second part of thetarget population of cells not directly producing the fusionpolypeptide.

[0014] Coupling products (including chemically coupled products) canalso be transported into a target population of cells by directlyexposing the cells to a preparation of the coupling products, thereby tocause the target cells to take them up.

[0015] In this specification, ‘VP22’ denotes: protein VP22 of HSV, e.g.of HSV1, and transport-active fragments and homologues thereof,including transport-active homologues from other herpesviruses includingvaricella zoster virus VZV, equine herpesvirus EHV and bovineherpesvirus BHV:; modified and mutant proteins and fusion polypeptidesand coupling products having homology therewith and a transport functioncorresponding to a transport function of VP22 of HSV1; and in contextalso relates to nucleic acid sequences encoding any of the above whetherin the form of naked DNA or RNA or of a vector, or of larger nucleicacid sequences including such sequences as sub-sequences.

[0016] Among sub-sequences of herpesviral VF22 protein with transportactivity we have found that for example transport activity is present inpolypeptides corresponding to aminoacids 60-301 and 159-301 of the fullHSV1 VP22 sequence (1-301). For the sequence, see e.g. FIG. 4 in WO97/05275. A polypeptide consisting of aa 175-301 of the VP22 sequencehas markedly less transport activity, and is less preferred inconnection with the present invention. Accordingly, the presentinvention relates in one aspect to coupled and fusion proteinscomprising a sub-sequence of VP22 containing a sequence startingpreferably from about aa 1-5 (or earlier, towards the N-terminal, in thenative VP22 sequence), to about aa301, and having (relative to the fullVP22 sequence) at least one deletion of at least part of the VP22sequence which can extend for example from the N-terminal to the citedstarting point, e.g. a deletion of all or part of the sequence of aboutaa 1-153. (Less preferably, such a deletion can extend further in theC-terminal direction, e.g. to about aa 175.) For example, partialsequences in the range from about aa 60-301 to about aa 159-301 areprovided.

[0017] VP22 sequences as contemplated herein extend to homologousproteins and fragments based on sequences of VP22 protein homologuesfrom other herpesviruses. e.g. the invention provides correspondingderivatives and uses of the known VP22-homologue sequences from VZV(e.g., all or homologous parts of the sequence from aa 1-302), from MDV(e.g. all or homologous parts of the sequence from aa 1-249) and fromEHV (e.g. all or homologous parts of the sequence from aa 1-258). Thesequences of the corresponding proteins from HSV2, VZV, BHV and MDV areavailable in public protein/nucleic acid sequence databases. Thus, forexample, within the EMBL/Genbank database, a VP22 sequence from HSV2 isavailable as gene item UL49 under accession no. Z86059 containing thecomplete genome of HSV2 strain HG52; the complete genome of VZVincluding the homologous gene/protein is available under accessionnumbers X04370, M14891, M16612; the corresponding protein sequence fromBHV is available as ‘bovine herpesvirus 1 virion tegument protein’ underaccession number U21137: and the corresponding sequence from MDV isavailable as gene item UL49 under accession number L10233 for ‘gallidherpesvirus type 1 homologous sequence genes’. In these proteins,especially those from HSV2 and VZV, corresponding deletions can be made,e.g., of sequences homologous to aa 1-159 of VP22 from HSV1. These citedsequences are hereby incorporated herein by reference. Homologiesbetween them are readily accessible by the use of standard algorithmsand software, for example those mentioned in WO 95/12573, page 9.

[0018] Furthermore, chimeric VP22 proteins and protein sequences arealso useful within the context of the present invention, e.g. a proteinsequence from VP22 of HSV1 for part of which a homologous sequence fromthe corresponding VP22 homologue of another herpesvirus has beensubstituted. For example, into the sequence of polypeptide 159-301 fromVP22 of HSV1, C-terminal sequences can be substituted from VP22 of HSV2or from the VP22 homologue of BHV.

[0019] It has been found that deletion of the 34-aminoacid C-terminalsequence from VP22 of HSV1 abolishes transport-activity, thus thissequence region contains essential elements for transport activity.According to a further aspect of the invention, there are providedcoupled and fusion polypeptides comprising the 34-aminoacid C-terminalsequence from VP22. or a variant thereof, together with a sequence fromanother protein selected from: (a) proteins for cell cycle control; (b)proteins that are conditionally cytotoxic or lethal upon administration(to a cell containing them) of a corresponding drug or activatorcompound, (c) antigenic sequences or antigenic proteins (e.g. of greaterthan 12 aminoacid residues in length) from microbial and viral antigensand tumour antigens, (d) immunomodulating proteins; and (e) therapeuticproteins. These are provided for example for use by administration inthe form of protein to cells that will take them up. Coupled products ofmodified terminal fragments having at least one mutation insertion ordeletion relative to the C-terminal 34 aminoacid sequence of HSV1 VF22are also provided.

[0020] It has also been found that sequences necessary for transportactivity contain one or a plurality of aminoacid sequence motifs ortheir homologues from the C-terminal sequence of VP22 of HSV1 or otherherpesviruses, which can be selected from RSASR, RTASR, RSRAR, RTRAR,ATATR, and wherein the third or fourth residue A can be duplicated, e.g.25 in RSAASR. Corresponding fusion polypeptides with proteins of thekinds mentioned herein are also provided.

[0021] In addition to their uses as indicated elsewhere herein, thecoupled and fusion polypeptides can also be used to raise antibodieswhich can be used in diagnostic and monitoring specific binding assaysin per-se known manner, e.g. for monitoring the intracellularlocalization of the coupled or fusion proteins themselves or theircomponents.

[0022] (‘VP22’ herein is not intended to include natural unmodified VP22protein or corresponding gene in its natural and unmodified associationwith herpes virus in its various natural lifecycle stages, e.g. inassociation with herpesvirus which has not been subjected to genomicalteration. However, ‘VP22’ does for example refer to the correspondingprotein or gene of a virus which has for example been altered in respectof its UL49/VP22 gene or function, or which has had inserted into itsgenome an additional and/or hybrid VP22 gene)

[0023] The coupling products or fusion proteins based on VP22 can have arange of molecular sizes. The products can in practice be for example upto about 70 kDa or more, e.g. 90 kDa or 100 kDa or more in respect ofthe size of the protein to be coupled or fused to VP22. The embodimentsof the invention include examples where the fusion peptide is e.g. atleast about 13 residues long, or more than about 12 aminoacid residueslong, e.g. other than a 12-residue antigenic epitope peptide. Theproteins to be fused can sometimes also be more than about 27 or 32 kDa,e.g. they can be other than 27 kDa in size. For example, one of theproteins that can be thus coupled, p53, itself has a size of about 53kDa. The coupled polypeptide or fusion protein, including the VP22component can have a size up to about 120 kDa, e.g. up to about 80 kDaor 100 kDa.

[0024] It is sometimes preferred that the VP22 sequence is fused at itsN-termninus to the sequence of the chosen other protein of one of thekinds mentioned herein. C-terminal fusions can sometimes becorrespondingly less preferred.

[0025] In the polypeptides of the invention, mutations of theconstituent aminoacid sequences (including those of the immunomodulatoryand other proteins mentioned herein) can be incorporated in the fusionpolypeptides and other coupled proteins. Included here are proteinshaving mutated sequences such that they remain homologous, e.g. insequence, function, and antigenic character or other function, with aprotein having the corresponding parent sequence. Such mutations canpreferably for example be mutations involving conservative aminoacidchanges. e.g. changes between aminoacids of broadly similar molecularproperties. For example, interchanges within the aliphatic groupalanine, valine, leucine and isoleucine can be considered asconservative. Sometimes substitution of glycine for one of these canalso be considered conservative. Interchanges within the aliphatic groupaspartate and glutamate can also be considered as conservative.Interchanges within the amide group asparagine and glutamine can also beconsidered as conservative. Interchanges within the hydroxy group serineand threonine can also be considered as conservative. Interchangeswithin the aromatic group phenylaalanine, tyrosine and tryptophan canalso be considered as conservative. Interchanges within the basic grouplysine, arginine and histidine can also be considered conservative.Interchanges within the sulphur-containing group methionine and cysteinecan also be considered conservative. Sometimes substitution within thegroup methionine and leucine can also be considered conservativePreferred conservative substitution groups are aspartate-glutamate:asparagine-glutamine, valine-leucine-isoleucine: alanine-valine:phenylalanine- tyrosine; and lysine-arginine. In other respects, mutatedsequences can comprise insertion and/or deletions. The mutated proteinsequences can additionally or alternatively be encoded bypolynucleotides that hybridize under stringent conditions with theappropriate strand of the naturally-occurring polynucleotide encodingthe parent protein, and can be tested for positive results in knownfunctional tests relevant to the parent protein. (‘Stringent conditions’are sequence dependent and will be different in different circumstances.Generally, stringent conditions can be selected to be about 5 deg C.lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and Ph) at which 50% of the target sequence hybridizes toa perfectly matched probe. Typically, stringent conditions will be thosein which the salt concentration is at least about 0.02 molar at pH 7 andthe temperature is at least about 60 dec C. As other factors may affectthe stringency of hybridization, including, among others, basecomposition and size of the complementary strands, the presence oforganic solvents and the extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one.)

[0026] Coupling with cell cycle control proteins:

[0027] In one useful class of embodiments of the invention, VP22 can becoupled with per-se known cell cycle control proteins. Thus, in anexample of the invention concerned with call cycle control, asparticularly described in an example below, VP22 can be coupled with p53protein. A purpose and use here can be to block cell cycle progression,especially in malignant cells.

[0028] VP22 can also usefully be coupled with cyclin-dependent kinnaseinhibitors, e.g. p15, p21 or p27. Normal cell cycle progression requiresthese proteins; absence of these can derepress the cell cycle, andcorresponding coupling products can be used for treatment of cancercells.

[0029] VP22 coupling products can also usefully be used in themodulation of apoptosis, e.g. to induce cell death, of the apoptosistype, by the introduction into a cell of a protein apoptic domaincoupled to VF,22, such as e.g. apoptosis protein bak, or its knownidentified apoptosis inducing peptide; or known related protein bad orbak. Here too the coupling product can be applied in the form either ofprotein or DNA encoding it. VP22 coupling products can be used in theform of VP22 with known proteins of the bcl2 family, such as bcl2itself, bcl-xl, or bclw, to mask or inhibit apoptosis where this isdesired, e.g in treatment of neuorodegeneration.

[0030] Other VP22 coupling products can be used to promote apoptosis,comprising VP22 linked with known ICE-like proteases. VP22 linkageproducts with inhibitors of ICE-like proteases, eg pseudosubstrates, canbe used to mask or overcome the apoptosis-stimulating effects theproteases themselves.

[0031] Thus, according to an embodiment of the invention there isprovided a fusion polypeptide comprising an aminoacid sequence with thetransport function of herpesviral VP22 protein and a sequence with thecell cycle control functionality of p53 protein. The fusion polypeptidecan include for example substantially the full length p53 sequence orsubstantially the full length VP22 sequence, or both.

[0032] Fusion with VP22 can thus be used for delivery of an agent forcell cycle control such as p53. (Where the description given hereinrefers to p53 and related peptides, it will be understood that, wherethe context admits, alternative cell cycle control agents, such as forexample those p53 analogues and other cell cycle control proteinsmentioned and referred to herein, are also contemplated, as are, moregenerally, alternative fusion or coupling partners for VP22, of any ofthe other types mentioned herein.) Once expressed in a subpopulation ofexpressing cells, such a fusion protein can be transported by the VP22transport mechanism from the expressing cell into a significantproportion of surrounding cells, and the foreign attached polypeptidecan then exert its functionality.

[0033] Also provided by this aspect of the invention are correspondingpolynucleotides, encoding a fusion polypeptide that comprises a sequencewith the transport function of herpesviral VP22 protein and a sequencewith the human/mammalian cell cycle-regulating function of p53 protein.The polynucleotide can be comprised in an open reading frame operablylinked to a suitable promoter sequence.

[0034] The polynucleotide can according to examples of the inventionform part of an expression vector, e.g. comprising the polynucleotidecarried in a plasmid. The expression vector can be for example a virusvector or a non-viral transfection vector. The vectors can for examplebe analogues or examples of those described and referred to in WO97/05255 or Elliott and O'Hare (1997).

[0035] Also provided by the invention are methods of inhibiting celldivision, which comprise exposing cells that have insufficientactive/free p53 to arrest their cell cycle, to contact with a fusionpolypeptide as described herein.

[0036] Among the methods of the invention is a method of inhibitingtumour cell division, which comprises exposing a tumour cell present ina tumour cell mass, the tumour cell comprising insufficient active/freep53 to arrest its cell cycle, to contact with a vector as describedherein, thereby causing the cell to express a fusion polypeptide asdescribed herein and to expose other cells of the tumour cell mass tocontact with the fusion polypeptide.

[0037] We have shown (see description below) that VP22-p53 can betransported to many untransfected cells in a monolayer. The fusionprotein can be functional in cell cycle arrest and/or induction ofapoptosis, for example both in primary expressing cells and in cellswhich have received VP22 via cell-to-cell spread. For example, thefusion protein can be applied to a p53 negative osteosarcoma cell lineSAOCS-2 (Diller et al., 1990). Functional p53 expressed in these cellscauses cell cycle arrest at the G₁-S boundary and ultimately cell deaththis can be assayed using confocal microscopy and antibodies againstspecific cell cycle markers. Function of the p53 fusion protein can alsobe used and assessed in other tumorigenic cell lines where p53 ispresent but contains specific and well characterized point mutationsleading to non-functionality.

[0038] A number of vector systems such as retroviral or adenoviralinfection or the injection of protein-liposome complexes can be readilyadapted to form examples of this invention for the administration ofcell-cycle control proteins to cells and tissues of human and non-humananimal subjects to be treated. For example, in relation to work on p53protein alone, these have clearly demonstrated that addition of wildtype p53 protein can curtail cancerous cell growth in vivo. A number oftherapeutic applications of non-invasive delivery of VP22 couplingproducts with cell-cycle control proteins will be apparent to theskilled reader.

[0039] For example, naked DNA for a VP22-protein fusion with a tumoureffector protein such as p53 can be injected into a tumour, e.g. a solidtumour, e.g. a solid tumor selected by molecular diagnostics for lack offunctional p53.

[0040] Recombinant viruses can be used as mentioned, encoding and ableto express VP22-p53 and equivalently-functioning fusion proteins. Forexample an adenovirus can express VP22-p53 and can be made dependent ona tumour-specific promoter to drive an essential viral gene such as E1a.More generally, a recombinant virus vector carrying such a fusion can bedefective, non-replicating or replication-restricted so that replicationis dependent on conditions prevailing in the target tissue or cell butnot in normal or non-target cells.

[0041] In certain examples of the invention, the protein having p53functionality can for example comprise variants or mutants of p53, forexample those variants as described in specification WO 97/04092 (RhonePoulenc Rorer S A: Bracco L, Conseiller E) (“New p53 variants e.g. witholigomerisation domain replaced by leucine zipper—useful for treatinghyper-proliferative disorders, especially cancer and restenosis”), whichdescribes inter alia the following variant proteins: (a) variants ofprotein p53 having at least part of the oligomerisation domain deletedand replaced by a leucine zipper domain, (b) variants of p53preferentially active in transformed cells, where all or part of atleast one functional domain has been deleted and replaced by aheterologous domain preferentially active in such cells; (c) variants ofp53 with a deletion in the C-terminal part, from residue 366, followedby a 19 amino acid sequence (encoded by a 76 bp fragment reproduced inthe specification) representing the last part of the alternativelyspliced part of murine p53; and (d) chimeric protein containing atransactivating domain, a DNA-binding domain, a nuclear localisationdomain and an oligomerisation domain, in which DNA-binding domain andthe nuclear localisation domain comprise amino acids 75-325 or 75-335 ofhuman wild-type p53.

[0042] In further examples of the invention, vectors and fusion proteinscan encode or comprise variant p53 polypeptides comprising chimaeric p53sequences including heterologous tetramerisation domains, which can beadapted from those described in specifications WO 96/15989 and U.S. Pat.No. 5,573,925 (Wistar Institute of Anatomy & Biology: Halazonetis T D)and used in corresponding ways. In such examples of the invention, thep53 sequences can comprise chimaeric p53 protein having a native p53sequence and 2 heterologous tetramerisation domain that formshomotetramers such that the resulting chimaeric protein cannothetero-oligomerise with wild-type or tumour derived mutant p53 and doesnot interfere with the native p53 tumour suppressing functionality.

[0043] Fusion proteins and vectors according to further examples of thepresent invention can be used for treatment of hyperproliferativedisease, especially cancer and autoimmune disease, e.g. restenosis, andparticularly for treatment of cells having a p53 mutation and which alsoexpress protein MDM2 at high level, including for example HPV-relatedcancer cells. They may also be used to kill hyperproliferating cells invitro. Such variants can involve active and stable tumour suppressor andapoptosis-inducing agents and are proposed to be active where the wildtype protein is not, i.e. not inactivated by dominant negative oroncogenic mutants, nor by other cellular proteins (because the leucinezipper domain prevents formation of inactive mixed oligomers).

[0044] Fusion proteins and vectors can also be used, according tofurther examples of the present invention, in medicaments forsuppressing neoplastic phenotype of cancer cells lacking wild-type p53protein in ways e.g. corresponding to the use of wild-type p35 gene asdescribed in specification EP 0 710 722 (Univ California: Chen P, LeeW), which describes genes and retroviral vectors for the purposes interalia of suppressing neoplastic phenotype in cancer cells such asosteosarcoma cells, lung carcinoma cells, colon carcinoma cells,lymphoma cells. leukaemia cells, soft tissue sarcoma cells or breast,bladder or prostate carcinoma cells.

[0045] Fusion proteins and vectors can also be used according to furtherexamples of the present invention, e.g. in ways corresponding to thosedescribed in specification WO 95/12560 (Univ Texas System: Roth J A et.al), which describes recombinant adenovirus which carries an adenovirusvector construct comprising an expression region encoding p53, and whichis capable of expressing the p53 in for example human malignant cells,and which can be used inter alia for regional delivery of tumoursuppressor gene p53, to diseased cells, either to restore p53 functionto p53 deficient cells, or to suppress tumour growth in cells havingabnormal p53 and thus to treat human malignancies such as breast andlung cancer. Such adenovirus may also be used for in vitro analyses andmutagenesis studies of various p53 genes.

[0046] Fusion proteins and vectors can also be used, according tofurther examples of the present invention as inhibitors of hepatitis Bvirus (HBV) replication, in ways corresponding to those described inU.S. Pat. No. 5,635,473 and WO 96/11017 (Mogam Biotechnology ResearchInstitute: H-S Lee et al).

[0047] Screening assays for identifying agents that effectively increasethe level of cell death, and which can act as p53 analogues and caninduce apoptosis in cells, are described for example in U.S. Pat. No.5,484,710 (La Jolla: J C Reed et al), particularly in example IV thereofAlso contemplated as alternative embodiments of the invention are fusionproteins and related materials incorporating VP22 functionality and Baxprotein functionality. In relation to Bax protein, reference is made toU.S. Pat. No. 5,484,710 and references cited therein, incorporatedherein by reference.

[0048] Coupling with ‘suicide protein’:

[0049] In a further class of embodiments of the invention, VP22 or afunctional sub-sequence thereof can be usefully coupled or fused withfor example a ‘suicide protein’ such as for example the known thymidinekinase, nitroreductase, or other enzyme or functional fragment thereofknown as applicable for a similar purpose. The coupling product canpenetrate into cells which are to be treated with (in the case ofthymidine kinase) ganciclovir or another drug (prodrug) of the samefamily, so that the prodrug is converted in the cells containing the‘suicide gene’ product to an active form to kill the cells.

[0050] Suitable examples of useful known suicide genes and correspondingpro-drugs are given and referred to for example in WO 94/13824 (UnivCurie Paris. M Caruso et al), in WO 95/05835 (Baylor College: S Chen etal), and in WO 93/08288 (Cancer Research Campaign Technology: G Anzelareet al), and WO 93/01231 (US DHHS; R M Blaese et al), and include,besides thymidine kinase (suicide gene) and ganciclovir/acyclovir(prodrug), nitroreductase (suicide gene) and CE1954 (prodrug), andcytosine deaminase (suicide gene) and 5-fluorcytosine (prodrug). Theseand other suicide proteins and corresponding (pro)drugs are alsoreviewed and their uses mentioned in ‘Genetic Prodrug ActivationTherapy’, A Rigg and K Sikora, Molecular Medicine Today, August 1997, pp359-366.

[0051] Where the VP22-TK fusion is presented in the form of DNA in anyof the ways described in WO 97/05265 or Elliott and O'Hare (1997), atarget cell can be transfected with the gene encoding this fusion, andthe expressed fusion can then be translocated out of the cell in whichit was expressed and into surrounding cells—producing a killing effecton such cells when treated with ganciclovir etc, an effect which isdifferent from, and can be additional to, known bystander effects.Alternatively, as with other embodiments, such a VP22-TK fusion can beapplied directly as protein.

[0052] Coupling with antigens:

[0053] In further embodiments, the invention concerns for exampletransport proteins related to VP22 or its active fragments fused infusion polypeptides or otherwise coupled with antigenic sequences orproteins (eg. of greater than 12 aminoacid residues in length) selectedfor example from any of the antigenic materials or other proteins andpeptides mentioned below.

[0054] In addition to the fusion polypeptides and coupling products, theinvention provides coupling hybrids comprising VP22 coupled to a DNAthat can for example comprise suitable known regulatory elements so thatit can be transcribed and translated, and containing an open readingframe encoding any of the proteins mentioned below.

[0055] Coupling with antigens VP22 can usefully be coupled with examplesof microbial and viral antigens and of tumour antigens such as thosementioned below.

[0056] Treatment with coupling products of VP22 involving antigens ofpathogens as provided hereby can evoke useful immune response againstcorresponding pathogens. Examples of such antigens are papilloma virusproteins L1 and L2. HIV proteins, gag, pol, env and nef, chlamydiaantigens (such as the chlamydia Major Outer Membrane Protein MOMP) andChlamydia heat shock proteins.

[0057] VP22 can also usefully be coupled with antigens from mycobacteriasuch as antigen from Mycobacterium tuberculosis.

[0058] Alternatively the antigen can be a tumour associated antigen,whereby the anti- tumour activity of the CTLs associated with tumourcell depletion is enhanced. It has been found that specific cytokinessuch as tumour necrosis factor-α, interferon gamma, interleukin-2,interleukin-4 and interleukin-7 are particularly useful in this regard.Tumour associated antigens and their role in the immunobiology ofcertain cancers is discussed for example by P van der Gruggen et al.,Current Opinion in Immunology, 4(5) (1992) 608-612. Particular examplesof such antigens which are envisaged for use in the contest of thepresent application are E6 and E7 antigens of human papillomavirus(especially for example of types 5, 11, 16, 18, etc.); Epstein-Barrvirus-derived proteins, e.g. those identified in references 24 and 25 inP van der Bruggen et al., cited above; antigens of the MAGE series asidentified in T. Boon, Adv Cancer Res 58 (1992), pp 177-210 and/or MZ2-Eand other antigens as identified in P. van der Bruggen et al, Science254 (1991) 1643-1647; melanoma proteins, e.g. human tyrosinase; andmucins, such as those identified in P. O. Livingston, in Current Opinionin Immunology 4 (:5) (1992) pp 624-629; e.g. MUC1 as identified in JBurchell et al. Int J Cancer 44 (1959) pp 591-696.

[0059] VP22 can also be usefully coupled with viral proteins such asglycoprotein antigens, e.g. from herpesviruses, such as gH or gD or gBof herpes simplex virus; or gp50 of pseudorabies virus, as an example ofan antigen of a veterinary pathogen, in this case a veterinary virus.

[0060] VP22 thus can be usefully coupled with antigens known from theprior art of malignant tumour treatment, including studies that havehighlighted the potential for therapeutic vaccination against tumoursusing autologous material derived from a patient's own tumour. Thetheory behind this approach is that tumour cells may express one or moreproteins or other biological macromolecules that are distinct fromnormal healthy cells, and which might therefore be used to target animmune response to recognise and destroy the tumour cells.

[0061] These tumour targets may be present ubiquitously in tumours of acertain type. A good example of this in cervical cancer, where the greatmajority of tumours express the human papillomavirus E6 and E7 proteins.In this case the tumour target is not a self protein, and hence itspotential as a unique tumour-specific marker for cancer immunotherapy isclear.

[0062] There is increasing evidence that certain self proteins can alsobe used as tumour target antigens. This is based on the observation thatthey are expressed consistently in tumour cells, but not in normalhealthy cells. Examples of these include the MAGE family of proteins. Itis expected that more self proteins useful as tumour targets remain tobe identified.

[0063] Tumour associated antigens and their role in the immunobiology ofcertain cancers are discussed for example by P van der Bruggen et al, inCurrent Opinion in Immunology, 4(5) (1992) 606-612. Other such antigens,of the MAGE series, are identified in T. Boon, Adv Cancer Res 511 (1992)pp 177-210, and MZ2-E and other related tumour antigens are identifiedin P. van der Bruggen et al, Science 254 (1991) 1643-1647:tumour-associated mucins are mentioned in P O Livingston, in CurrentOpinion in Immunology 4 (5) (1992). pp 624-629: e.g. MUC1 as mentionedin J Burchell et al, Int J Cancer 44 (1989) pp 691-696.

[0064] Coupling with immunomodulating proteins:

[0065] Embodiments of the invention of use in immune modulation includefor example the following. VP22 can usefully be coupled with examples ofcytokines or of other immunomodulatory compounds as mentioned below.Thus, VP22 can also be usefully coupled with immuno modulating proteins,e.g. those which enhance the immune response including the cytokine,interleukin 1, interleukin 2 and granulocyte-macrophage colonystimulating factor (GM-CSF). Such products can ,for example be used inways analogous to those mentioned in for example WO 96/26257 or WO97/14808, to alter, e.g. to increase, an immune response specific to atarget cell type, e.g. a tumour cell type, which has been exposed to theproduct either in-vitro or in-vivo.

[0066] As used herein, the expression “immunomodulatory protein” andrelated terms includes a protein or proteins which either enhance orsuppress a host immune response to a mutant virus or protein encodedthereby, or to an antigen such as an immunogen from a pathogen or sourceexogenous to the virus, or a tumour-associated antigen. Theimmunomodulating proteins are not normally those proteins presently usedas immunogens (antigens) in themselves. An immunomodulatory protein canbe a natural member of a human or non human animal immune system, e.g.of a mammalian immune system, with a functional binding capacity foranother natural constituent of such an immune system. Alternatively animmunomodulatory protein can be a protein encoded by a pathogen, whichhas a functional binding capacity for a natural constituent of such animmune system.

[0067] Alternatively an immunomodulatory protein can be an artificialprotein, for example a fragment of a natural immunomodulatory protein ora mutein of such a protein or fragment, or a fusion proteinincorporating any of these. Many immunomodulatory proteins, and geneticmaterials encoding them, and their nucleotide and aminoacid sequences,are known to the literature of this subject, and available in geneticsequence databases such as the EMBL database, and several arecommercially available in the form of engineered genetic material forcloning and other manipulation.

[0068] Immunomodulating proteins coupled with VP22 as described hereincan usefully for example be of sequences native to the species which isto receive treatment with these coupling products or with DNA e.g. inthe form of recombinant viruses, e.g. an immunomodulating protein ofhuman type for treatment of a human subject.

[0069] Examples of useful known immunomodulating proteins in thisconnection include cytokines, chemokines, complement components, immunesystem accessory and adhesion molecules and their receptors of human ornon-human animal specificity. Useful examples include GM-CSF, IL-2,IL-12, lymphotactin, CD40, and CD40L. Further useful examples includeinterleukins for example interleukins 1 to 15, interferons alpha, betaor gamma, tumour necrosis factor, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSFgranulocyte colony stimulating factor (G-CSF), chemokines such asneutrophil activating protein (NAP), macrophage chemoattractant andactivating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1and MIP-1b, complement components and their receptors, or an accessorymolecule such as B7.1, B7.2, ICAM-1, 2 or 3 and cytokine receptors.

[0070] OX40 and OX40-ligand (OX40L) (gp34) (see e.g. WO 95/12573, WO95/21251 and WO 21915) are further useful examples of immunomodulatoryproteins. Immunomodulatory proteins can for various purposes be of humanor non-human animal specificity and can be represented for presentpurposes, as the case may be and as may be convenient, by extracellulardomains and other fragments with the binding activity of the naturallyoccurring proteins, and muteins thereof, and their fusion proteins withother polypeptide sequences, e.g. with immunoglobulin heavy chainconstant domains. Where nucleotide sequences encoding more than oneimmunomodulating protein are inserted, they can for example comprisemore than one cytokine or a combination of cytokine(s) andaccessory/adhesion molecule(s).

[0071] Immune response evoked by the use of such VP22 coupling productsor by vectors encoding them can include immune responses of a variety oftypes, e.g. a response against a virally-encoded protein, and/or aresponse against a host antigen, being a response stimulated by a viralvector or by the expression of a heterologous gene encoded thereby, e.g.the coupling product with VP22. Among the uses of the mutant virusvectors as described herein is e.g. to protect a subject of asusceptible species against infection by a corresponding wild-type viruswhen the subject is treated therewith, e.g. infected therewith, e.g. bydirect immunisation.

[0072] An immunomodulatory protein to be coupled with VP22 can be itselfa hybrid or fusion protein which comprises a polypeptide region havinghomology to and functionality of an immunomodulatory protein, linked toa polypeptide region having another homology and optionally anotherfunctionality. For example, the immunomodulatory protein can becomprise, or correspond in functionality to the gp34 protein identifiedas a binding partner to human Ox-40 (see W Godfrey et al, J Exp Med180(2) 1994 pp 757-702, and references cited therein, including S Miuraet al, Mol Cell Biol 11(3) 1991, pp 113-132). The version of thisprotein functionality that can be encoded in the mutant viral genome cancorrespond to the natural gp34 sequence itself, or to a fragmenttherefore to a hybrid expression product e.g. based on the (C terminal)extra cellular (binding) domain of gp34 fused to another protein, e.g.to the constant region of an immunoglobulin heavy chain such as humanIgG1, e g. with the extracellular domain of gp34 (a type 2 membraneprotein) fused at its N-terminal to the C-terminal of the immunoglobulinconstant domain.

[0073] Others of the immunomodulatory proteins can also be carried andexpressed in such derivative and hybrid forms, including mutated formsas mentioned herein.

[0074] In certain examples the immunomodulating protein can comprise acytokine, preferably granulocyte macrophage colony stimulating factor(GM-CSF), e.g. murine or preferably human GM-CSF.

[0075] Murine and human GM-CSFs are both known: the murine GM-CSF geneencodes a polypeptide of 141 amino acids, the mature secretedglycoprotein having a molecular weight of between 14 k-30 k daltonsdepending on the degree of glycosylation. GM-CSF generically is a memberof the haematopoietic growth factor family and was first defined andidentified by its ability to stimulate in vitro colony formation inhaematopoietic progenitors. GM-CSF is a patent activator of neutrophils,eosinophils and macrophage-monocyte function, enhancing migration,phagocytosis, major histocompatibility complex (MHC) expression, andinitiating a cascade of bioactive molecules which further stimulate theimmune system. GM- CSF is currently being clinically evaluated fortreatment of neutropenia following chemotherapy and as an adjuvant incancer therapy.

[0076] The heterologous nucleotide sequence employed can comprise aheterologous gene, gene fragment or combination of genes provided itencodes an immunomodulation protein as defined above.

[0077] According to examples of the invention, combinations of two ormore immunomodulatory proteins can be used for the purposes describedherein, in particular examples, given for illustration only and notlimitation, combinations involving IL2, GMCSF, lymphotactin and/or CD40Lcan be used with each other or with others of the immunomodulatoryproteins cited above Each of the other binary combinations of theimmunomodulatory proteins mentioned above are also given by, and withinthe scope of, this disclosure.

[0078] Other coupling products:

[0079] In certain embodiments the invention can be useful in genetherapy applications: thus, for example, VP22 can also be usefullycoupled with examples of genes used or proposed to be used in-genetherapy including: the gene for human adenosine deaminase (ADA), asmentioned in for example WO 92/10504 (K W Culver et al: US Secretary forCommerce & Celico Inc), WO 89/12109 & EP 0 420 911 (I H Pastan et al):the cystic fibrosis gene and variants described in WO 91/02796 (L-C Tsuiet al: HSC Research & University of Michigan), in WO 92/05273 (F SCollins & J M Wilson: University of Michigan) and in WO 94/12649 (R JGregory et al: Genzyme Corp).

[0080] VP22 can also usefully be coupled with known transcriptionalregulatory proteins such as NF-AT, which becomes activated bytranslocation to the nucleus and induces transcription of interleukine.g. of IL1. The coupling with VP22 can be used here to avoid retentionof the coupled product in the cytoplasm.

[0081] The invention also includes coupled and fusion proteins in whicha linker sequence is provided that enables the fusion protein to besplit intracellularly to enable separation of the antigenic part, suchas that mentioned above, from the transport protein part. Acleavage-inducing sequence can comprise for example the aminoacidsub-sequence RVCSNPPCETHETGTTNTATATSN or other cleavage sequencesindicated for example in A C Wilson et al, in Genes and Development 9(1995) 2445-2453.

[0082] Also provided by the present invention are processes for treatingcells with coupling products as described herein, so as to produceimmunogenic, immunomodulatory, cytotoxic/lethal or therapeutic effects.

[0083] Examples of materials and processes as described herein areuseful in the modulation of cellular activity, e g. with the aim andeffect of producing or altering immune responses, for example for theprophylaxis or therapy of disease, e.g. the production of immuneresponses against pathogens or tumours.

[0084] Other uses for certain of the materials and processes hereofinclude the regulation of gene expression in calls. e.g. for purposes ofcorrective gene therapy and/or for reducing or controlling tumour cellgrowth and activity. Cell treatments according to the invention can bein-vitro, ex-vivo or in-vivo.

[0085] Among the derivatives of VP22 that can be used according toaspects of the invention as transport active substances and for couplingwith materials to be transported, for the purposes set forth elsewhereherein, are peptides comprising a transport-active functional sequencefrom the C terminal section of VP22.

[0086] Non-limitative examples of treatment methods using the materialsdescribed herein comprise treatment of antigen-presenting cells or cellpreparations containing them with a fusion of VP22 and an antigen, e.g.one of those antigens mentioned above, (or with a vector, e.g. a viralvector, encoding such a fusion), so as to procure processing of theantigen and presentation by the MHCl route so as to procure a CTLresponse to the antigen. The methods so provided include priming andexpansion of T cells and adoptive immunotherapy using the materials soobtained, in a manner otherwise analogous to known priming, expansionand adoptive immunotherapy methods.

[0087] A number of vector systems such as retroviral or adenoviralinfection or the injection of protein-liposome complexes, as well asherpesviral vector systems, can be readily adapted to form examples ofthis invention. For example, naked DNA for a VP22-protein fusion with aprotein so one of the kinds mentioned herein can be injected into atissue to be treated, according to the nature and purpose of the proteinto be delivered. Recombinant viruses can be used as mentioned, encodingand able to express VP22 fusion proteins. A recombinant virus vectorcarrying such a fusion can be defective, non-replicating orreplication-restricted so that replication is dependent on conditionsprevailing in the target tissue or cell but not in normal or non-targetcells.

[0088] Vectors and fusion proteins of examples of the invention can beuseful in gene therapy, and to treat or protect against abnormal cellproliferation, esp. cancer but also psoriasis, atherosclerosis andarterial restenosis, and to induce apoptosis of e.g. proliferatinglymphocytes, i.e. to induce tolerance, e.g. to prevent transplantrejection or for treatment of autoimmune diseases such as systemic lupuserythematosus or rheumatoid arthritis.

[0089] In addition to medical therapeutic applications, the effect shownherein can also be exploited by assays, provided by the invention, whichrely on substrate-enzyme interactions or the interaction of proteinsexpressed in different cellular populations.

[0090] An embodiment of the invention is further described, withoutintent to limit the invention, with reference to the accompanyingdrawings and to the materials and methods described below.

[0091] In the accompanying drawings,

[0092]FIG. 1 illustrates that:

[0093] Mock-transfected cos-1 cells were labelled by indirectimmunofluorescence with antibodies for VP22 (FIG. 1a), p53 (FIG. 1c) andthe CMV epitope (FIG. 1d) to establish the levels of background labelCells transfected with pc49epB (FIG. 1b) and labelled for P22demonstrate a typical VP22 cytoplasmic pattern with clear spread to thenuclei of adjacent cells. Cells transfected with the VP22-p53 fusionprotein construct p4955ep+10 were labelled for VP22 and p53 (FIGS. 1eand 1 f) or VP22 and epitope (FIGS. 1g and h): the fusion protein can bedetected in the nuclei of cells adjacent to the primary expressing cell.

[0094]FIG. 2 is a plasmid map to illustrate p4953ep+10, encoding afusion protein comprising sequences VP22, p53 and an epitope tag.

[0095]FIG. 3 illustrates that

[0096] Protein extracts from cos-1 cells transfected with a range ofplasmid constructs were analysed by western blot The panel shownleftmost has been probed with an antibody against VP22 and demonstratesthat pUL49epB and pc49epB plasmids encoding VP22 alone generate aprotein of 38kDa, the VP22-p53 fusion protein expressed from p4953ep+10produces a protein of approx. 50 kDa with very little degradation.

[0097] The panel shown rightmost has been probed with an antibodyagainst p53 and demonstrates that cells transfected with plasmidsencoding either p53 alone (pcB6+p53) or the p4953ep+10 fusion proteinconstruct produce p53 protein at 53 kDa. The p4953ep+10 construct alsosynthesises the VP22-p53 fusion protein at 90 kDa, the p53 in thissample may be a degradation product or more likely endogenously inducedp53.

MATERIALS AND METHODS

[0098] Cell culture and transfection

[0099] Cos-1 cells were grown in Dulbecco's modified MEM supplementedwith 10% new born calf serum at 37° C. with 5% CO₂.

[0100] Transfections were performed using the BES/CaCl₂ method (Elliottand O'Hare, 1997) with 200 ng test plasmid with 1800 ng pUB19.Transfections were allowed to proceed for 48 h at which point themonolayers were harvested for immunofluorescence or western blotanalysis.

[0101] Immunofluorescence and antibodies

[0102] Cell monolayers on coverslips were fixed with 100% methanol for15 mins at room temperature and labelled as described in Elliott andO'Hare (1997). All antibodies were diluted in PBS+10% serum. VP22 wasdetected using a rabbit polyclonal antibody AGV30 (1:500), p53 wasdetected using a mouse monoclonal antibody DO-1 (Santa-Cruz Ltd), theCMV epitope was detected using a mouse monoclonal antibody CMV LNA(Capricorn Ltd). images were obtained using a Bio-Rad MRC500 confocalmicroscope.

[0103] Plasmid Constructs

[0104] The VP22-p53 fusion protein construct was generated by cloning afull length p53 PCR fragment C-terminal to VP22 into a unique Bam site,keeping both VP22 and the CMV epitope in frame.

[0105] Western blot analysis

[0106] Western blots were prober with anti-VP22 (1:10,000),anti-p53.(1:1000).

[0107] We constructed an epitope-tagged full length in-frame VP22-p53fusion protein construct (FIG. 2). This vector generates a fusionprotein of approx 90 kDa when expressed in Cos-1 cells, with very littleprotein degradation as judged by western blot analysis (FIG. 3). Whentested for delivery by intercellular trafficking, the fusion proteinappears to function exactly as VP22 alone. It is located in thecytoplasm of primary transfected cells as shown by immunofluorescenceusing methanol-fixed Cos-1 cell monolayers labelled with anti-VP22(FIGS. 1e and 1 g), p53 (FIG. 1f) or -epitope (FIG. 1h) antibodies andis able to move very efficiently into the nuclei of neighbouring cells.The relative efficiency of transport has not been empirically determinedbut appears only slightly less than VP22 alone.

[0108] In further experiments, p53-negative osteosarcoma cells weretransfected (using the calcium phosphate technique) with naked DNAexpressibly encoding either (a) wild-type VP22, (b) wild-type p53) or(c) the VP22-p53 fusion protein described above. The transfected cells(b) and (c) showed ability to undergo apoptosis, unlike the controlcells (a), indicating that the VP22-p53 fusion protein retains thefunctionality of p53.

[0109] In variants of the example given here, VP22 deletion constructswith decreased fusion protein size can be made if desired. e.g. toimprove rate or extent of transport, and without loss of proteinfunction.

[0110] In further variants, the order of the components of the fusioncan be varied, e.g. the p53 and VP22 sequences can readily be includedin the order opposite to the order involved in the plasmid shown in FIG.2, with satisfactory results.

[0111] The present disclosure extends to modifications and variations ofthe description given herein inclusive of the attached claims that willbe apparent to the reader skilled in the art. The disclosure hereof,incorporating WO 97/05265 and of Elliott and O'Hare (1997) which aremade an integral par hereof, is intended to extend in particular toclasses and subclasses of the products and generally to combinations andto subcombinations of the features mentioned, described and referred toin the disclosure. Documents cited herein, including the referencesbelow, are hereby incorporated in their entirety by reference for allpurposes.

[0112] Additional References

[0113] Diller, L., Kassel, J. Nelson, C. E., Gryka, M. A., Litwak, G.,Gebhardt, M. Bressac, B., Ozturk, M., Baker, S. J., Vogelstein, B. andS. H. Friend, (1990) p53 functions as a cell cycle control protein inosteosarcomas. Mol. Cell. Bio. 10:5772-5781.

[0114] Elliott G. and P. O'Hare (1997) Intercellular trafficking andprotein delivery by a herpesvirus structural protein. Cell 82:223-233.

[0115] Ensoli, B.. Buonaguro, L., Barillari, G., Fiorelli, V.,Gendelman, R., Morgan, R. A., Wingfield, P. and R. C. Gallo, (1993)Release , uptake and effects of extracellular human immunodeficiencyvirus Tat protein on cell groves and viral transactivation. J. Virol.67:277-287.

[0116] Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L.,Pepinsky, B. and J. Barsoum. (1994) Tat-mediated delivery ofheterologous proteins into cells. Proc. Natl. Acad. Sci. 91:664-668,

[0117] Hamada, K., Alemany, R., Zhang, W-W, Hittelman, W. N., Lotan, R.,Roth, J. A. and M. F. Mitchell. (1996) Adenovirus-mediated transfer of awild-type p53 gene and induction of apoptosis in cervical cancer. CancerResearch 56:3047-3054.

[0118] Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough R.and T. Maciag. (1992) Heat shock induces the release of fibroblastgrowth factor 1 from NIH3T3 cells. Proc. Natl. Acad. Sci.89:10691-10695.

[0119] Kumar, X, M., Srinivas, S., Detolla, L. J., Yu S.F., Stass, S. A.and A. J. Mixson. (1997) Parenteral gene therapy with p53 inhibits humanbreast cancer tumors in vivo through a bystander effect without evidenceof toxicity. Hum-Gene Therapy 8:177-185.

[0120] Levine, A. J. (1997) p53, the cellular gatekeeper for growth anddivision. Cell 88:323-331.

[0121] Nielsen, L. L., Dell, J., Maxwell, E., Armstrong, L., Maneval, C.and J. J. Catino, (1997) Efficacy of p53 adenovirus-mediated genetherapy against human breast cancer xenografts. Cancer Gene Therapy4:129-138.

[0122] Sandig, V., Brand, K., Herwig, S., Lukas, J., Bartek, J. and M.Strauss. (1997) Adenovirally transferred p16 and p53 genes cooperate toinduce apoptotic tumor cell death. Nature Med. 3:313-319.

[0123] Zheng, P.S., Iwasaka. T., Ouchida, M., Fukuda, K., Yokoyama, M.and H. Sugimori. (1996) Growth suppression of a cervical cancer cellline (TMCC-1) by the human wild type p53 gene. Gynecol Oncol.60:245-250.

1 11 1 5 PRT Artificial Sequence Description of Artificial Sequencepeptide 1 Arg Ser Ala Ser Arg 1 5 2 6 PRT Artificial SequenceDescription of Artificial Sequence peptide 2 Arg Ser Ala Ala Ser Arg 1 53 5 PRT Artificial Sequence Description of Artificial Sequence peptide 3Arg Thr Ala Ser Arg 1 5 4 6 PRT Artificial Sequence Description ofArtificial Sequence peptide 4 Arg Thr Ala Ala Ser Arg 1 5 5 5 PRTArtificial Sequence Description of Artificial Sequence peptide 5 Arg SerArg Ala Arg 1 5 6 6 PRT Artificial Sequence Description of ArtificialSequence peptide 6 Arg Ser Arg Ala Ala Arg 1 5 7 5 PRT ArtificialSequence Description of Artificial Sequence peptide 7 Arg Thr Arg AlaArg 1 5 8 6 PRT Artificial Sequence Description of Artificial Sequencepeptide 8 Arg Thr Arg Ala Ala Arg 1 5 9 5 PRT Artificial SequenceDescription of Artificial Sequence peptide 9 Ala Thr Ala Thr Arg 1 5 106 PRT Artificial Sequence Description of Artificial Sequence peptide 10Ala Thr Ala Ala Thr Arg 1 5 11 24 PRT Artificial Sequence Description ofArtificial Sequence linker sequence 11 Arg Val Cys Ser Asn Pro Pro CysGlu Thr His Glu Thr Gly Thr Thr 1 5 10 15 Asn Thr Ala Thr Ala Thr SerAsn 20

1. Coupled polypeptides and fusion polypeptides comprising (i) anaminoacid sequence with the transport function of herpesviral VP22protein and (ii) another protein sequence selected from (a) proteins forcell cycle control; (b) suicide proteins (proteins that areconditionally cytotoxic or lethal upon administration, to a cellcontaining them, of a corresponding (pro)drug or activator compound):(c) antigenic sequences or antigenic proteins (e.g. of greater than 12aminoacid residues in length) from microbial and viral antigens andtumour antigens; (d) immunomodulating proteins; and (e) therapeuticproteins.
 2. A polypeptide according to claim 1 where said other proteinsequence is from a mammalian (e.g. human) cell cycle control protein. 3.A polypeptide according to claim 2 where said other protein sequence isfrom a mammalian (e.g. human) protein for increasing or inducing cellapoptosis or for conferring on 2 cell the ability to undergo apoptosis.4. A polypeptide according to claim 2 where said other protein sequenceis from a mammalian (e.g. human) cell cycle control protein selectedfrom p53 protein, cyclin dependent kinase inhibitors, and proteins oilthe bcl2 and bax families.
 5. A polypeptide according to claim 4, whichis a fusion polypeptides and comprises a sequence from a p53 protein. 6.A fusion polypeptide according to claim 5, comprising substantially afull length VP22 sequence and substantially a full length p53 sequence.7. A polypeptide according to claim 1 where said other protein sequenceis from a suicide protein.
 8. A polypeptide according to claim 7 wheresaid suicide protein is selected from thymidine kinase andnitroreductase.
 9. A polypeptide according to claim 7, comprisingsubstantially the full length VP22 sequence.
 10. A polypeptide accordingto claim 1, which is a fusion polypeptide, and comprises acleavage-inducing linker sequence located between the VP22 sequence andsaid other protein sequence.
 11. A polypeptide according to claim 1,comprising a sub-sequence of HSV VP22 starting from about aa 159 andextending to about aa 301, and having (relative to the full VP22sequence) at least one deletion of at least part of the VP22 sequenceextending for example from the N-terminal to the sequence of about aa1-158.
 12. A polypeptide according to claim 1, which comprises asequence corresponding to aminoacids 60-301 or 159-301 of the full HSVVP22 sequence.